Eyster, H. Clyde

The Ohio Journal of Science

KnowledgeBank at OSU

Ohio I. Sci. NUTRITIVE LEVELS IN STRIPMINE PLANTSBRIEF NOTENUTRIENT CONCENTRATION REQUIREMENTS FORCHLORELLA SOROKTNIANA1CLYDE EYSTER, Mobile College, Mobile, AL 3(5013OHIO J. SCI. 7S(2): 79, 1978Minimum, optimal and maximal con-centration of essential nutrient elementsfor the growth of Chlorella sorokinianawas determined. Growth measurementsincluded optical density and packed cellvolumes.Culturing apparatus: A photothermo-stat of the type described by Myers(1950) was constructed. The interiordimensions were 22 inches long by 3inches wide by 73^ 2 inches deep and theplastic Lucite aquarium lid accommo-dated 22 test tubes held snugly in place.Water at 38.8° C was continuouslycirculated by a ^3 nP electric pump. Afilter inserted between the pump and theaquarium kept the water clear. Thetemperature controller was adjusted sothat a temperature of 39° C was main-tained in the aquarium bath and theaverage operating temperature was foundto be 38.8° C, with a standard deviationof ±0.16° C.The culturing apparatus was equippedwith 2 banks of lights, a bank for eachside of the aquarium bath. Each bankhas three 48-inch Sylvania cool whitepower tubes, providing the tube culturesa light intensity of 925 ft-candles fromeach side.Agitation and carbon dioxide for thetest tube cultures was provided by passinginto each tube culture a mixture of 5%1Manuscript received January 19, 1977 and inrevised form as a note May 19, 1977 (#77-8).CO2-Air through a glass bubbling tube.Each bubbling tube was 20 cm long andwas constricted approximately 3 cm be-low its upper end in order to hold a smallcotton plug. The bubbling tube wasfitted into the top of the test tube bymeans of a cotton plug wrapped withgauze. A paper collar 5 cm in length wasmade to extend above the cotton plug toprotect the culture from microbial con-tamination, when the bubbling tubewas elevated for optical density measure-ments.Two manifolds, each providing 12 out-lets, were constructed from brass tubingand needle valves. Autoclaved surgicalrubber tubing connected the needle valveoutlets with the bubbling tube of eachtest tube culture. The 5%CO2 - Air,was humidified by passing it through asterilized 300 ml side-arm bubbling flaskcontaining 100 ml of distilled water, andthen directed into the manifolds whichwere cleaned and steamed before beinginstalled.Culture procedure: Cultures were grownin triplicate in modified Knop's medium(Eyster 1966; 1967), which was modifiedto contain urea, micro-nutrient trace ele-ments, and ethylene diamine tetraacetate(EDTA) as a chelating agent. Modifi-cations of Knop's medium for determin-ing elemental nutrient requirements areshown in table 1.It was found necessary to use triple80 CLYDE EYSTERTABLE 1Knop' s Medium modifications for determining the elemental nutrient requirements ofChlorella sorokiniana.Vol. 78Element*MagnesiumSulfur**Potassium***PhosphorusNitrogenIronCalciumZincManganeseCopperDeletedMagnesium sulfateMagnesium sulfatePotassium dihydrogenphosphatePotassium dihydrogenphosphateUrea, nitratefFerric nitrateCalcium chlorideTEM*TEM°TEM*SubstitutedSodium sulfateMagnesium chlorideSodium dihydrogenphosphatePotassium chloride———TEM minus Zinc chlorideTEM minus ManganesesulfateTEM minus CoppersulfateConcentration RegulatorMagnesium sulfateSodium sulfatePotassium chlorideSodium dihydrogenphosphate—Ferric nitrateCalcium chlorideZinc chlorideManganese sulfateCopper sulfate*Amount contributed by inoculum was included in calculating element levels.**Trace elements ordinarily supplied as sulfates were supplied as chlorides.***pH was adjusted to 6.8 with NaOH instead of KOH. The disodium salt of EDTA was usedinstead of its potassium salt.fOr other nitrogen sources.•Trace element mixture: manganese, copper, molybdenum, cobalt salts and zinc chloride.°Trace element mixture: zinc, copper, molybdenum, cobalt salts and manganese sulfate.*Trace element mixture: manganese, zinc, molybdenum, cobalt salts and copper sulfate.glass distilled water because the singledistilled water had about 50 ppb copperions. Analyses for copper by the use ofzinc dibenzyldithiocarbamate in CC14(Stone et al 1953) revealed that KH2PO4had appreciable (88 mg/mole) copper asa contaminant, and that MgSO4-7H2O,CaCl2-2H2O, and H3BO3 had smalleramounts (42 mg/mole, 56 mg/mole, and2 mg/mole, respectively). KNO3 wasfound to have only 0.8 mg copper permole compared with 4800 mg per equiva-lent of urea. This perhaps explains whyWalker (1953) could demonstrate a cop-per requirement with nitrate in the me-dium but not with urea in the medium.Copper deficiency symptoms are morepronounced at relatively high levels ofEDTA. It is recommended that copperessentiality efforts include, first, the puri-fication of water, KH2PO4, MgSO4, CaCl2,urea, and H3BO3 for copper removal, andsecond, an increase in concentrationlevels of EDTA.For my studies, 4.5 ml of culturemedium were placed in each test tube.A glass bubbling tube or "stem", fittedwith a 5 cm long paper collar and a cot-ton plug, was inserted into each test tube,which was then steam-sterilized in anautoclave at 10 lb pressure for 10 minutes.Sterile urea (0.25 ml) and bacteria-freeinoculum (0.25 ml) were then added toeach cooled tube culture by means of aheat-sterilized 1.0 ml B-D tuberculinsyringe fitted with a long sterile needle.This needle was inserted through thehollow center of the stem to deliver, in 2operations, the urea and the inoculum.It was customary to prepare the mediumso that 4.5 ml contained the nutrients in-tended for 5.0 ml. This technique al-lowed the addition of 0.25 ml of sub-cultured inoculum and 0.25 ml of sterileurea solution (Eyster 1966; 1967).A bacteria filter apparatus with afine fritted disc was used to producebacteria-free solutions of urea. An elec-tric instrument sterilizer was utilized toheat-sterilize the syringes and needles.Tubes of trypticase soy broth and tubesof nutrient agar were used for the purposeof checking the sterility of the C. soro-kiniana cultures periodically.Experimental measurements: Growth atthe end of 23 hours was measured by ob-taining absorbance readings at 550 m/x.Optical absorption by C. sorokiniana cellsOhio j . Sci. CHLORELLA. GROWTH NUTRIENTSTABLE 2Summary of mineral nutrient requirements for the growth ofNutrientElementNitrogenMagnesiumSulfurPotassiumPhosphorusCalciumIronZincManganeseCopperEDTAChlorella sorokiniana.MinimumConcentration1 x 10~3M1 x 1(T5M1 x 1CT6M1 x 10-f>M5 x 1()-SM1 x 1()-5M1 x 1()-6M1 x 10-°M3 x Kr 'M1()-12MzeroOptimumConcentration1 x 10"2M to 5 x l(r2M1 x 10~4M to 4 x 10~2M2x 10-"M t o 5 x Kr2M5 x 1()-4M to 1 x 1()-'M5 x 1()-4M to 2 x Kr2M5 x 10~4M to 3 x 1()-3M2 x 1(TBM to 1 x 10"3M1 x 10~6M to 1 x Kr2M1 x 1CT5M to 1 x 10~2M1()-8M to 10-3M3 x 10"4M to 2.5 x 1()-3MMaximumConcentration2 x lO^M1 x lO^M1 x K)-iM5 x Kr'M1 x Kr'M1 x Kr2M2 x 10~3M—2 x 10~3M4.5 x 10"3M81did not follow Beer's Law at densitiesabove 0.850 and direct absorption read-ings of dense cultures were less than thecalculated total absorption values de-rived from the absorption of dilutedaliquots. It was our practice to place0.50 ml of dense culture in 9.50 ml offresh medium, read the absorbance ofthe mixture, and then multiply the ab-sorbance value by 20, giving a totalabsorbance value for the dense culture.The most dense cultures had to be diluted40-fold. Additional measurements in-cluded pH and packed cell volume de-terminations (Eyster 1966; 1967).A summary of the elemental nutrientrequirements for the growth of C.sorokiniana is given in table 2, which isbased on the data reported by Eyster(1966; 1967), and in a specially preparedmanuscript (Eyster 1976) with com-plete supportive data. Nutrient ele-ments which were found to be requiredwere N, P, Mg, S, K, Fe, Ca, Mn, Zn,and Cu. On the contrary, there was noindication that B, Mo, Na, Co, Sr, andCl were required.Acknowledgments. The research reported inthis paper was sponsored by the EnvironmentalSciences Division, USAF School of AerospaceMedicine, Aerospace Medical Division, AFSC,United States Air Force, Brooks AF.B, Texas,under Contract No. AF 41 (009)-2414 withDayton Laboratory of Monsanto ResearchCorporation.LITERATURE CITEDEyster, H. C. 1966 Mineral nutrient re-quirements of Chlorella sorokiniana in con-tinuous pure culture. Final Report. USAFSchool of Aerospace Medicine, AerospaceMedical Division (AFSC). Brooks Air ForceBase, Texas. 186 pp.1967 Mineral nutrient requirementsof Chlorella sorokiniana in continuous pureculture. Technical Report SAM-TR-67-40.USAF School of Aerospace Medicine, Aero-space Medicine Division (AFSC). BrooksAir Force Base, Texas. 53 pp.1976 Optimal nutrient concentrationrequirements for Chlorella sorokiniana. Photo-copied manuscript with 14 large tables ofdata. Available from the author or theMobile College Library, Mobile, Alabama36613. 40 pp.Myers, J. 1950 The culture of algae for phys-iological research. In: Brunei, F., G. W.Prescott and L. H. Tiffany (eds.), TheCulturing of Algae, Charles F. KetteringFoundation, Yellow Springs, OH. p. 45-51.Stone, I., R. Ettinger and C. Gantz 1953 Non-ashing technique for determination of tracesof copper in malt beverages. Anal. Chem.25: 893-895.Walker, J. B. 1953 Inorganic micronutrientrequirements of Chlorella. I. Requirementsfor calcium (or strontium), copper, andmolybdenum. Arch. Biochem. 46: 1—11.

Brief Note  Nutrient Concentration Requirements for Chlorella Sorokiniana

https://kb.osu.edu/bitstream/1811/22516/1/V078N2_079.pdf

Brief Note Nutrient Concentration Requirements for Chlorella Sorokiniana

Abstract

Similar works

Full text

Available Versions

KnowledgeBank at OSU